Estelle Anceaume

Metrology
Metrology : Science of measurements. Measurement of parameters to know if the microscope is in
optimal condition.

Today we will only look at some measurements and settings:

 Laser or fluorescent lamp power measurement

 Laser or lamp alignment
 Field homogeneity

There are other measures:

 PSF
 Co-alignment
 Stability (X, Y, Z)

Power measurement : Allows to check the stability of the laser or lamp. This measurement is
important for intensity quantification experiments.

For example: To know if a protein is more expressed according to the experimental conditions.

 Material needed: Power meter (Thorlabs)

 In a day, there may be small variations in intensity. Diode and Led are stable in time
 Tip: Mix up the experimental conditions during a microscopy session. Do not do all the
control conditions on one day and the test conditions on another day.
 If very low power: Laser alignment / Change the optical fiber / Change the light source.

Laser alignment: readjustment of lenses positions in the optical path of the laser bench to obtain
maximum power.

 Material needed : Power meter (Thorlabs)

 Call the manufacturer or After Sales Service

Field homogeneity : Allows to check if the light source is homogeneous in the field.

 Material needed : Slide tétra-spectre / Fiji pluging « MetroloJ »

 To be checked for all objectives, excitation lines and filters.
 If inhomogeneity : Center the fluorescent lamp / Change the optical fiber / Check the
position of the filters and dichroics / Contact SAV

PSF : A simple way to characterize the resolution power in the x, y, and z dimensions.

 Material needed : Slides containing 170 nm fluorescent beads / Z-stack acquisition / Fiji
pluging « MetroloJ »

Co-alignment : Check if the two excitation lines are well co-aligned

 Material needed : Slides containing multi-color fluorescent beads / Z-stack / Fiji pluging
« MetroloJ »
 Important for co-localisation experiments.
 Check the position of the filters / Use multi-band dichroics / Check the state of the lens
(default of the chromatic or optical aberration correction).
Estelle Anceaume

Stability (X, Y, Z) : Allows to check the stability of the stage to avoid drifts in X, Y and Z

 Materials needed : Slide of fluorescent beads / Tme-lapse acquisition

 Check that the stage is well fixed / Change the stage / Contact SAV.

References :

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9526251/ : Quality assessment in light microscopy

for routine use through simple tools and robust metrics. RTMFM-2022

https://imagejdocu.list.lu/plugin/analysis/metroloj/start : MetroloJ Pluging RTMFM

https://www.youtube.com/watch?v=ll4X_e8_mo8 : PSF video RTMFM.